Novel uses of adipic acid

ABSTRACT

The present invention relates to a method for preventing or treating obesity, diabetes, dyslipidemia, fatty liver or insulin resistance syndrome, comprising: administering to a mammalian subject in need thereof a therapeutically effective amount of a composition comprising adipic acid. The present composition containing adipic acid as an active ingredient is very effective in preventing or treating obesity, diabetes, dyslipidemia, fatty liver (preferably, non-alcoholic fatty liver) or insulin resistance syndrome. The composition of the present invention useful as pharmaceuticals compositions or functional food compositions has therapeutic efficacies for obesity, dyslipidemia or fatty liver, and also induces significant decrease in fasting glucose level and blood insulin level to improve type 2 diabetes, insulin resistance and related metabolic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C.§119 (a), this application claims the benefit ofearlier filing date and right of priority to Korean Patent ApplicationNo. 10-2011-0052770 filed on Jun. 1, 2011, the contents of which arehereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel uses of adipic acid, inparticular, a method for preventing or treating obesity, diabetes,dyslipidemia, non-alcoholic fatty liver or insulin resistance syndrome.

2. Background of Technique

At present, 1.7 billion people amounting to about 25% of the worldpopulation are overweight (BMI>25) and more than 300 million peopleincluding 120 million in the US, Europe and Japan are classified asobese (BMI>30). Among the OECD countries, the US has the highest obesityrate of 31% of population, followed by Mexico (24%), England (23%),Greece (22%), Australia (22%), New Zealand (21%), Hungary (19%), Canada(14%), Spain (13%), Ireland (13%), Germany (13%), Portugal (13%),Finland (13%), Turkey (12%) and Belgium (12%). The number of obesepeople in China is 70 million and the body weight control-related marketis expanding, estimated at about 10 billion yuan. Childhood obesity isalso increasing rapidly worldwide, with 1 in 5 children being obese. Assuch, childhood obesity is becoming a serious social issue. Sincechildhood obesity is the main cause of the life style diseases includingdiabetes, hypertension, stroke, etc. with increased blood cholesteroland triglyceride level, 80% or more of obese children are likely tobecome obese adults. Further, since increased fat stimulates secretionof sex hormones and induces early adolescence, childhood obesity maycause growth problems. Also, it negatively affects blood circulation andnourishment.

According to statistics of Korea Institute for Health and SocialAffairs, the socioeconomic loss caused by obesity and obesity-relatedcomplications in 2006 is estimated at 2.1 trillion won including medicalcost and indirect cost such as loss of earning. Thus, in 2010, theKorean government has decided to reduce the obesity rate down to 20% inadults and to 15% in youth, and is exploring ways to accurately defineand diagnose obesity and metabolic diseases. To strategies for achievingobjective, the government has been looking for the exact definition andmeasuring method about obesity and metabolic diseases.

Obesity drugs that are marketed inside and outside Korea include‘Xenical’ (Roche Korea) with orlistat as main ingredient and approved bythe FDA, Reductir (Ilsung Pharmaceuticals) with sibutramine as mainingredient, ‘Exolise’ (Guju Pharma) with green tea catechol as mainingredient, or the like. Xenical, which reduces absorption of fat byinhibiting lipase, has the gastrointestinal-related side effects such assteatorrhea, gas generation and reduced absorption of oil-solublevitamins. Reductil, which increases serotonin and noradrenaline levelsin the sympathetic nervous system, has side effects such as headache,dry mouth, loss of appetite, insomnia, constipation, etc. Besides, alarge number of anti-obesity drugs have been withdrawn from the marketdue to severe side effects. For example, aminophylline is reported tohave various side effects in the nervous, circulatory and digestivesystems despite its excellent effect of reducing body fat. Also,fenfluramine, dexfenfluramine, topiramate, ephedrine, etc. have beenbanned from being marketed as obesity drugs. As the synthetic drugs showlimitations in side effects and in overcoming chronic diseases, foodsand drugs derived from natural sources are drawing attentions.

NAFLD (non-alcoholic fatty liver disease) refers to a liver diseaseassociated with triglyceride accumulation in the liver regardless ofdrinking alcohols, including steatosis and NASH (non-alcoholicsteatohepatitis). Steatosis is considered benign diseases with goodprognosis in clinic, but NASH accompanied with fatty liver, inflammationor fibrosis is recognized as progressive liver diseases inducingcirrhosis or liver cancer.

The obesity and the insulin resistance are representative of riskfactors for NAFLD. The risk factors of hepatic fibrosis progression are,for example, obesity (BMI>30), ratio of liver function parametersdetected in serum (AST/ALT>1) and diabetes. Hepatitis C carriers havingthe non-alcoholic fatty liver would be progressed to liver cancer; andtherefore there are emerging needs for prevention and treatment ofnon-alcoholic fatty liver. 69-100% of patients with non-alcoholic fattyliver have obesity and 20-40% of patients in obesity are accompaniedwith fatty liver. Especially, prevalence of liver disease in the maleobesity patients is higher than obesity women patients. There has beenreported that 3-30% of adults with normal weight as well as obesitypatients have fatty liver diseases in the Western society. In Japanhaving similar diet patterns to Korea, the prevalence of non-alcoholicfatty liver is estimated to be approximately 20% and 1% of them isestimated to be NASH. Non-alcoholic fatty liver became problems in obesechildren as well as adults. 10-77% of obese children (inhabited inEurope, USA and Asia) show non-alcoholic fatty liver lesions, becausethe most important risk factor for non-alcoholic liver disease isobesity.

The pathogenesis of non-alcoholic fatty liver may be explained by twomechanisms. The first mechanism is that the increase in free fatty acidsinhibits fatty acid oxidation in hepatocytes, thereby accumulating fattyacids in hepatocytes to cause non-alcoholic fatty liver. The secondmechanism involves a variety of physiological factors associated withinflammation and fibrosis progression. More specifically, the increasein levels of fatty acid induces to elevate the expression of cytochromeperoxidase 2E1 and CYP2E1 and to generate reactive oxygen speciesresulting in lipid peroxidation of liver cell membrane and the increasein LPS and oxidative stress, increasing the level of TNF-α beingresponsible for apoptosis of hepatocytes, finally inducing liver damage.The insulin resistance and the accumulation of fatty acids contribute tomitochondrial dysfunction, and the latter increases reactive oxygenspecies and nitric oxide synthase (NOS), thereby inducing cell death.

The best way to treat NAFDL may be considered a weight loss throughlifestyle changes (e.g., the exercise). However, when it is difficult totreat NAFDL with only exercise, chemotherapeutics may be combined withexercise. Chemotherapeutics for non-alcoholic fatty liver areclassified: First, there are drugs with remediation of risk factors fortreating and improving fatty liver, including obesity drugs (e.g.,orlistat), insulin resistance drugs (e.g., metformin, pioglitazone androsiglitazone), and hyperlipidemia drugs (e.g., clofibrate, gemfibrozil,bezafibrate, atorvastatin and simvastatin). Metformin increasesoxidation of fatty acids, decreases lipogenic enzymes and improveshyperinsulinism and insulin resistance. Meanwhile, thiazolidinedione,rosiglitazone and pioglitazone are capable of activating PPARγ asnuclear hormone receptors to promote the glucose uptake in muscles.Secondly, there are drugs with potentials to recover liver cell damagebeing independent on remediation of risk factors of non-alcoholic fattyliver, including hepatocyte protectors (e.g., ursodeoxycholoc acid andtaurine), antioxidants (e.g., Vitamins E and C), and nutritionalsupplements (e.g., lecithin, betaine, and N-acetylcysteine).Unfortunately, there are no more plausible drugs with excellenttherapeutic effects without adverse effects.

Adipic acid contained in sugar beet, beetroot and gambir is carboxylicacid compound, which is called as 1,4-butanedicarboxylic acid,1,6-hexanedioic acid, acifloctin, acinetten, adipinic acid, hexanedioicacid, octafluorohexanedioic acid or molten adipic acid. Adipic acid hasthe molecular formula of C₆H₁₀O₄ and a molecular weight of 146.1 g/mol,represented by the following chemical formula:

Adipic acid has been registered as “acidity regulator” in the foodadditives database of Domestic Food Code. It is also registered asacidity regulator in the Food and Color Additive Database of the U.S.FDA. In addition, it has been also reported to have anti-ketogeniceffect and anti-hypertensive effect. In the rat, adipic acid is known tobe effective for improving ketosis caused by long-chain (long-chain) andshort-chain monocarboxylic acids (Mortensen PB, Biochim Biophys Acta664:335˜48, (1981)). Furthermore, a clinical study conducted in South Korearevealed that patients treated with amlodipine adipate for 8 weeksexhibited alleviated symptoms of hypertension (Lee H Y. Et al., ClinicalTherapeutics., 27728-739, (2005)).

Adipic acid is an edible substance with a significant high safety. It isnoteworthy that the reported LD₅₀ of adipic acid is more than 11,000mg/kg (rats) and 4,175 mg/kg (mice), respectively (Kennedy G L Jr, DrugChem Toxicol. 25 (2) :191-202, (2002)).

Throughout the specification, a number of publications and patentdocuments are referred to and cited. The disclosure of the citedpublications and patent documents is incorporated herein by reference inits entirety to more clearly describe the state of the related art andthe present disclosure.

SUMMARY OF THE INVENTION

The present inventors have made intensive studies to develop naturalsubstance or compounds derived from natural substance having therapeuticefficacies for metabolic diseases including obesity, diabetes,dyslipidemia and non-alcoholic fatty liver. As a result, they have foundout that adipic acid is significantly effective in prevention ortreatment of the metabolic diseases.

Accordingly, it is an object of this invention to provide a compositionfor preventing or treating obesity, diabetes, dyslipidemia,non-alcoholic fatty liver or insulin resistance syndrome.

It is another object of this invention to provide a method forpreventing or treating obesity, diabetes, dyslipidemia, non-alcoholicfatty liver or insulin resistance syndrome.

Other features and aspects will be apparent from the following detaileddescription, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 represent changes of body weight gain (g) in mice fed withtest diets over time (x axis represents maintenance day) (FIG. 1),accumulated body weight gain for 10 weeks (g/70 days) (FIG. 2) and dailyfood intake (g/day) (FIG. 3), respectively. The results are representedas mean±SEM (standard error) of values obtained from eight mice. Thecharacters above the bars indicate significant difference among dietarygroups by one-way analysis of variance (ANOVA) test and Duncan'smultiple range test (P <0.001).

FIG. 4 shows visceral fat-pad weight (g) of mice fed with test diets.The results are represented as mean±SEM (standard error) of valuesobtained from eight mice. The characters above the bars indicatesignificant difference among dietary groups by one-way analysis ofvariance (ANOVA) test and Duncan's multiple range test (P <0.001).

FIG. 5 represents images of visceral fat tissues of mice fed with testdiets.

FIGS. 6 and 7 show liver weight (g) and liver lipid level of mice fedwith adipic acid. The results are represented as mean±SEM (standarderror) of values obtained from eight mice. The characters above the barsindicate significant difference among dietary groups by one-way analysisof variance (ANOVA) test and Duncan's multiple range test (P <0.05).

FIG. 8 represents the expression profiles of genes relating to adipocytedifferentiation in mice visceral fat tissues. The left panel representsimages of RT-PCR analysis results for PPARγ2, C/EBPα, and aP2, and theright panel represents relative expression levels of these genes. Thedata were normalized to GAPDH mRNA levels and expressed as relativevalues to those from ND mouse. The results are represented as mean±SEM(standard error) of values obtained from eight mice. The charactersabove the bars indicate significant difference in dietary groups byone-way analysis of variance (ANOVA) test and Duncan's multiple rangetest (P <0.05).

FIG. 9 shows change of gene expression profiles of pro-inflammatorycytokines in mice visceral fat tissues.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, there is provided a compositionfor preventing or treating metabolic diseases such as obesity, diabetes,dyslipidemia, non-alcoholic fatty liver and insulin resistance syndrome.

In another aspect of the present invention, there is provided a methodfor preventing or treating metabolic diseases such as obesity, diabetes,dyslipidemia, non-alcoholic fatty liver and insulin resistance syndromecomprising: administering to a mammalian subject in need thereof atherapeutically effective amount of a composition comprising adipicacid.

The present inventors have made intensive studies to develop naturalsubstance or compounds derived from natural substance having therapeuticefficacies for metabolic diseases including obesity, diabetes,dyslipidemia and non-alcoholic fatty liver. As a result, they have foundout that adipic acid contained in diverse plants is significantlyeffective in prevention or treatment of the metabolic diseases.

According to the present invention, adipic acid is responsible fordecrease of visceral fat-pad weights and plasma lipids such astriglyceride, total cholesterol and free fatty acid as well as plasmaglucose level, and is capable of inducing significant change in lipidlevel of liver tissues and liver function parameters.

According to the present invention, adipic acid induces decreases inbody weight, visceral fat, levels of plasma lipids such as triglyceride,total cholesterol and free fatty acid, and triglyceride level of livertissues, thereby considerably alleviating obesity induced by HFD(high-fat diet). Furthermore, the gene expressions of both nucleartranscription factors and their target gene which are increased byobesity induced with HFD, are decreased by adipic acid administrationand the gene expressions of TNF-α and IL-6 are also decreasedsignificantly to attenuate inflammatory responses in progressivenon-alcoholic fatty liver.

As used herein, the term “fatty liver” refers to a condition where fataccumulates excessively in liver cells due to the disorder of lipidmetabolism. It may cause various diseases such as angina, myocardialinfarction, stroke, arteriosclerosis and pancreatitis.

As used herein, the term “diabetes” refers to a chronic diseasecharacterized by relative or absolute lack of insulin, leading toglucose intolerance. As used herein, the term diabetes includes allkinds of diabetes, such as type 1 diabetes, type 2 diabetes and geneticdiabetes. Type 1 diabetes, which is insulin-dependent diabetes, mainlyresults from the destruction of β-cells. Type 2 diabetes, which isnon-insulin-dependent diabetes, is caused by insufficient secretion ofinsulin after meals or by insulin resistance.

As used herein, the term “insulin resistance” refers to a condition inwhich the natural hormone insulin becomes less effective at loweringblood sugars. When insulin resistance is high, the human body createstoo much insulin to result in developments of not only hypertension anddyslipidemia but also heart diseases and diabetes. Especially, in type 2diabetes, the increase in insulin is unrecognized in muscle and fattissue, such that insulin action does not occur.

As used herein the term “insulin resistance syndrome” refers to ageneral term for disease which is induced by insulin resistance. It ischaracterized by cell resistance against insulin action,hyperinsulinemia, increase of very low density lipoprotein (VLDL) andtriglyceride, decrease of high density lipoprotein (HDL) andhypertension. The insulin resistance syndrome is usually considered as arisk factor for cardiovascular disease and type 2 diabetes (Reaven GM,Diabetes, 37:1595-607 (1988)). In addition, it has been reported thatinsulin resistance increases intracellular oxidative stress togetherwith risk factors such as hypertension, diabetes and smoking, and alterssignal transduction to cause inflammatory responses, such thatatherosclerosis is developed (Freeman BA. Et al., Lab Invest. 47:412-26, (1982)), Kawamura M. et al, J Clin Invest. 94: 771-8 (1994)).

As used herein the term “metabolic diseases” refer to a group of a widevariety of diseases caused by risk factors for various cardiovasculardiseases and type 2 diabetes, including insulin resistance and itsrelated diverse and complicated metabolic and clinical abnormalities. In1988, Reaven suggested that a common cause of these symptoms is insulinresistance and named insulin resistance syndrome; however, in 1998, WHOnewly introduced the term “metabolic syndrome or metabolic diseases”,because insulin resistance may not explain all the elements of thesesymptoms.

The composition of the present invention comprising adipic acid as anactive ingredient has activity for preventing diverse diseases ofmetabolic diseases such as obesity, diabetes, hyperlipemia,non-alcoholic fatty liver or insulin resistance syndrome. Thecomposition of the present invention may prevent or treat metabolicdiseases by its various action mechanisms.

As used herein the term “hyperlipidemia” refers to a disease caused byhigher level of blood lipids due to poor metabolism of lipids such astriglyceride and cholesterol. More specifically, hyperlipidemia ischaracterized by increased levels of lipids such as triglyceride, LDLcholesterol, phospholipids and free fatty acids in blood, includinghypercholesterolemia and hypertriglyceridemia.

According to a preferred embodiment, the insulin resistance syndrometreated by the present invention comprises obesity, hypertension,atherosclerosis, hyperlipidemia, hyperinsulinemia, non-alcoholic fattyliver and type 2 diabetes.

According to a preferred embodiment, the composition of the presentinvention decreases levels of blood fat, liver fat or visceral fat.

The term “liver” or “visceral” is used to encompass organ, tissue andcell.

As addressed in Examples, the groups fed with the composition of thepresent invention showed significantly reduced lipid level in blood andliver tissues, and the total visceral fat weight was significantlyreduced by 21% as compared to HFD.

According to a more preferred embodiment, the fat reduced by the presentinvention comprises triglyceride, cholesterol and free fatty acid.

According to a more preferred embodiment, the visceral fat reduced bythe present invention comprises epididymal fat, perirenal fat,mesenteric fat and/or retroperitoneal fat.

According to a preferred embodiment, the composition of the presentinvention decreases activity of ALT (alanine aminotransferase) or AST(aspartate aminotransferase).

ALT and AST as indicators for liver function are enzymes exhibitingincreased levels in blood upon damage of liver.

The composition of the present invention significantly reduced both ALT(by 40%) and AST (by 27%) in the blood as compared to HFD-fed groups,addressing that adipic acid has the excellent efficacies of improvingfatty liver, preferably non-alcoholic fatty liver.

According to a preferred embodiment, the composition of the presentinvention decreases the expression of PPARγ (Peroxisome proliferatoractivated receptor gamma), C/EBPs (CCAAT enhancer-binding proteins) oraP2 (fatty acid binding protein). The composition of the presentinvention decreases the expression of not only PPARγ and C/EBPs but alsoaP2 as their target gene, finally decreasing amount of visceral fat. TheaP2 gene is expressed during preadipocyte proliferation anddifferentiation to adipocytes and its expression is regulated by PPARγand C/EBPs.

According to a preferred embodiment, the composition of the presentinvention decreases the expression of TNF-α (tumor necrosisfactor-alpha) and IL-6 (interleukin-6).

It has been reported that when obesity is induced by HFD, free fattyacids (especially saturated fat) in body fluids are increased. The freefatty acids as ligands bound to TLR4 activate IKK and then NF-κB, andstimulate the secretion of pro-inflammatory cytokines such as TNF-α andIL-6 to cause inflammatory response.

In addition, TNF-α and IL-6 activate both the cytokine signaling 3(SOCS3) and JNK and induce phosphorylation of serine residues of insulinreceptor substrates (IRS) to inhibit glucose transport, finally causinginsulin resistance in peripheral tissues of liver or muscle.

According to a preferred embodiment, the composition of the presentinvention decreases glucose level in blood.

As demonstrated in Examples, the composition of the present inventionsignificantly reduced fasting blood sugar level (by 25%) to improveinsulin resistance, thereby exhibiting therapeutic efficacies formetabolic diseases.

According to a preferred embodiment, adipic acid used in the presentinvention is derived from plants. Adipic acid may be extracted orfractionated from plants such as Betavulgaris var (Sugar beet), Daucuscarota var (Beetroot) and Uncaria gambir (Gambir). Adipic acid may beextracted from several plants or chemically synthesized in a massproduction manner.

According to a preferred embodiment, the composition of the presentinvention is a pharmaceutical composition.

When the composition of the present disclosure is prepared as apharmaceutical composition, the pharmaceutical composition of thepresent disclosure may comprise a pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier included in the pharmaceuticalcomposition of the present disclosure is one commonly used in thepreparation of formulations and includes lactose, dextrose, sucrose,sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate,gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,mineral oil, etc., but is not limited thereto. The pharmaceuticalcomposition of the present disclosure may further include, in additionto the above-described components, a lubricant, a wetting agent, asweetener, a fragrance, an emulsifier, a suspending agent, apreservative, or the like. Suitable pharmaceutically acceptableexcipients and formulations are described in detail in Remington'sPharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present disclosure may beadministered orally or parenterally. Preferably, it may be administeredparenterally.

An appropriate administration dosage of the pharmaceutical compositionof the present disclosure may be determined variously depending on suchfactors as preparation method, administration method, age, body weightand gender of a patient, pathological condition, diet, administrationtime, administration route, excretion rate or response sensitivity.Specifically, a daily dosage of the pharmaceutical composition of thepresent disclosure may be 0.001-100 mg/kg.

The pharmaceutical composition of the present disclosure may be preparedinto a unit dosage form or multiple dosage form along with apharmaceutically acceptable carrier and/or excipient according to amethod that can be easily employed by those skilled in the art. Theformulation may be in the form of solution in oily or aqueous medium,suspension, syrup, emulsion, extract, dust, powder, granule, tablet orcapsule, and may further include a dispersant or stabilizer.

According to a preferred embodiment, the composition of the presentinvention is a food composition.

When the composition of the present disclosure is prepared as a foodcomposition, the food composition of the present disclosure maycomprise, in addition to adipic acid of the present disclosure as theactive ingredient, ingredients commonly added for preparation of food.For example, proteins, carbohydrates, fats, nutrients, seasoning orflavors may be added. The carbohydrate may be, for example, a sugar suchas a monosaccharide, e.g. glucose, fructose, etc., a disaccharide, e.g.maltose, sucrose, oligosaccharide, etc. or a polysaccharide, e.g.dextrin, cyclodextrin, etc. or a sugar alcohol such as xylitol,sorbitol, erythritol, etc. The flavor may be a natural flavor[thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.]) ora synthetic flavor (saccharin, aspartame, etc.).

For example, when the food composition of the present disclosure isprepared as a drink, it may further comprise, in addition to adipic acidof the present disclosure as the active ingredient, citric acid,high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruitjuice, eucommia extract, jujube extract, licorice extract, or the like.

The features and advantages of the present invention may be summarizedas follows:

-   -   (a) The present composition containing adipic acid as an active        ingredient is very effective in preventing or treating obesity,        diabetes, dyslipidemia, fatty liver or insulin resistance        syndrome.    -   (b) Adipic acid as active ingredients for the present        composition may be isolated from various plants and chemically        synthesized.    -   (c) The composition of the present invention useful as        pharmaceuticals compositions or functional food compositions has        therapeutic efficacies for obesity, diabetes, hyperlipidemia or        fatty liver, and also induces significant decrease in fasting        glucose level and blood insulin level to improve type 2        diabetes, insulin resistance and related metabolic diseases.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES Example 1 Reduction of Body and Visceral Fat-Pad Weights byAdipic Acid 1) Preparation of Test Diets and Maintenance of Test Animals

The obesity-inducing control diet used in the test was high-fat diet(HFD: 40% fat calorie, 17 g lard+3% corn oil/100 g diet). Dietssupplemented with adipic acid (adipic acid-supplemented high fat diet,NED) had the same composition as HFD, except that adipic acid wasincluded at 0.2% (see Table 1). The normal diet group (Chow) was fedwith commercially available rodent chow. Adipic acid was purchased fromSigma-Aldrich.

TABLE 1 Compositions of test diets adipic acid-supplemented High-fatdiet (HFD) diet (ADD) Ingredients (g/kg diet) (g/kg diet) Casein 200 200D/L-Methionine 3 3 Corn starch 111 109 Sucrose 370 370 Cellulose 50 50Corn oil 30 30 Lard 170 170 Vitamin complex 12 12 Mineral complex 42 42Choline bitartrate 2 2 Cholesterol 10 10 Tert-butylhydroquinone 0.040.04 adipic acid — 2 Total (g) 1,000 1000 Fat (% calorie) 39.0 39.0Total calorie (kJ/kg diet) 19,315 19,3155-week-old male C57BL/6J mice (Orient, Korea) were accustomed to thelaboratory environment for 1 week while feeding solid feed. Then, theywere randomly divided into normal diet, high-fat diet and test groupsand bred for a total of 10 weeks. The diet was given between 10 and 11a.m. every day together with water. Food intake was measured every dayand body weight was measured once a week. In order to avoid transientbody weight increase after feed intake, body weight was measured 2 hoursafter removing the feed. After fasting the test animal for at least 12hours and anesthetizing with diethyl ether, blood, liver and visceralfat (epididymal fat, perirenal fat, mesenteric fat and retroperitonealfat) were taken and weighed after washing with 0.1 M PBS (pH 7.4). Bloodtaken from the abdominal aorta was centrifuged at 1000×g for 15 minutesfor the separation of plasma.

2) Changes of Body and Visceral Fat-Pad Weights

After feeding the test diet for 10 weeks, the adipic acid-supplementedgroup showed significant decrease of body weight gain by 49% as comparedto HFD. The dietary supplementation with adipic acid induced nosignificant change in daily food intake (FIG. 3). Therefore, it would beunderstood that the body weight-decreasing effect by adipic acid is notdue to the suppression of appetite.

After feeding the test diet for 10 weeks, the epididymal, perirenal,mesenteric, and retroperitoneal fat-pads contained in the visceral fatwere removed and weighed. The adipic acid-supplemented group showedsignificantly reduced weights of the epididymal, perirenal, mesenteric,and retroperitoneal fat-pads as compared to HFD. The total visceral fatweight was significantly reduced by 21% in the AAD group than in the HFDgroup

(FIGS. 4 and 5). Accordingly, it would be appreciated that adipic acidhas excellent effects to reduce body weights and visceral fat-padweights.

Example 2 Prevention and Treatment of Hyperlipidemia and Type 2 Diabetesby Adipic Acid 1) Biochemical Analysis of Blood

After 10 weeks of breeding, total cholesterol, triglyceride and glucoselevels in the plasma and lipid levels in the liver tissue were measuredas follows. Total cholesterol, triglyceride, free fatty acid and glucoselevels in the plasma were measured twice for each using a commerciallyavailable kit (Bio Clinical System). The activity of ALT (alanineaminotransferase) and AST (aspartate aminotransferase) used as liverfunction indicator were measured twice for each using a commerciallyavailable kit (Bio Clinical System, Korea).

2) Changes of Plasma Lipid, Glucose Level and Liver Function Level

After feeding the test diet for 10 weeks, the adipic acid-supplementedgroup showed significantly lower plasma levels of triglyceride level(26%), total cholesterol level (24%), and free fatty acid level (32%) ascompared to the HFD group (Table 2). Moreover, adipic acid supplementedto the HFD resulted in a significant reduction of the fasting bloodsugar level by 25%, as compared to HFD. The HFD group exhibitedsignificantly higher plasma activities of ALT (alanine aminotransferase)and AST (aspartate aminotransferase), which are parameters for hepaticfunction, as compared to the normal diet group and and the AAD groupshowed significantly decreased plasma activities of ALT (by 40%) and AST(by 27%) as compared to HFD group. Accordingly, it could be recognizedthat adipic acid has the excellent effects of improving hyperlipidemia,fasting blood sugar level, and hepatic function parameters in obesityinduced by HFD.

TABLE 2 Biochemical indicator related obesity in blood of mice fed withadipic acid Normal diet group Adipic acid — (Chow) High fat diet (HFD)(AAD) Triglyceride (mmol/L) 0.50 ± 0.08^(b) 1.21 ± 0.12^(a) 0.89 ±0.05^(b) Total cholesterol (mmol/L) 1.56 ± 0.03^(b) 3.53 ± 0.32^(a) 2.66± 0.41^(b) Free fatty acid (uEq/L) 513 ± 48^(b)  1312 ± 128^(a)  890 ±56^(c)  Glucose (mmol/L) 5.87 ± 0.95^(b) 8.79 ± 0.53^(a)  6.6 ± 0.41^(b)ALT (IU/L)  9.3 ± 0.86^(b) 13.1 ± 0.27^(a)  7.9 ± 0.78^(c) AST (IU/L) 8.4 ± 0.66^(b) 12.3 ± 1.03^(a)  8.9 ± 0.28^(b)The characters above the bars indicate significant difference with otherdietary groups by one-way analysis of variance (ANOVA) test and Duncan'smultiple range test (P <0.05).

Example 3 Prevention and Treatment of Non-Alcoholic Fatty Liver byAdipic Acid 1) Analysis of Lipid Level in Liver Tissues

Lipids were extracted from the liver tissue according to Folch et al.'smethod Folch J et al., J Biol Chem. 226: 497-509 (1957)). After adding 1mL of distilled water to 0.25 g of liver tissue, the liver tissue washomogenized using a Polytron homogenizer (IKA-Werke GmbH & Co.,Ultra-Turrax, Staufen, Germany). After adding 5 mL ofchloroform:methanol solution (2:1, v/v) to the homogenate and mixingwell, the mixture was centrifuged at 1000×g for 10 minutes. After adding2 mL of chloroform:methanol solution (2:1, v/v) again to thesupernatant, the same procedure was repeated to completely separate thelipid components of the liver. After adding 3 mL ofchloroform:methano1:0.05% CaCl2 (3:48:47, v/v/v) solution to theremaining pellets and mixing well for 1 minute, followed bycentrifugation at 1000×g for 10 minutes, the resulting pellets werecompletely dried with nitrogen gas. The dried lipids were dissolved in 1mL of methanol and then analyzed.

The same kit (Bio Clinical System) as that used for the plasma analysiswas used to measure the triglyceride and cholesterol levels of the livertissue.

2) Changes of Hepatic Lipid Levels

After feeding the test diet for 10 weeks, liver absolute weight (g) inthe adipic acid-supplemented group was significantly reduced by 12%, ascompared to the HFD group (FIG. 6). After feeding the test diet for 10weeks, the adipic acid-supplemented group showed significantly decreasedlevels of triglyceride (by 26%), cholesterol (by 44%) and free fattyacid (by 37%) in liver tissue as compared to HFD (FIG. 7). Accordingly,it could be understood that adipic acid has the excellent effect ofsignificantly improving fatty liver in obesity induced with high-fatdiet.

Example 4 Regulation of Gene Expressions in Visceral Fat Tissues byAdipic Acid 1) RNA Extraction and RT-PCR (ReverseTranscription-Polymerase Chain Reaction) Analysis

After adding 1 mL of Trizol agent per 0.1 g of visceral fat tissues, themixture was homogenized and centrifuged at 12,000×g for 10 min at 4° C.The supernatant was transferred to a new tube and 200 μl of chloroformwas added to the tube, followed by vortexing. The same procedure wasrepeated twice and then the supernatant was transferred to a new tube,followed by addition of isopropanol and the supernatant at 1:1 ratio.The mixture was vigorously shaken 10 times and then incubated for 10 minat room temperature, followed by centrifugation at 12,000×g for 10 minat 4° C. to remove the supernatant. After adding 1 mL of 70% ethanol tothe remaining pellet, it was centrifuged at 7,500×g for 5 min at 4° C.After removing the ethanol, the RNA pellet contained in the tube wasdried for 5 min at 4° C. and dissolved in nuclease-free water. The RNAsample concentration was measured at a wavelength of 260 nm and 280 nmusing a UV/VIS spectrophotometer (Beckman coulter, DU730) and theintegrity of RNA sample was verified by agarose gel electrophoresis.

The RNA sample obtained from the visceral fat tissues was trasnscribedusing oligo dT primer and SuperScript reverse transcriptase (GIBCO BRL,Gaithersburg, Md., USA) to synthesize cDNA. The PCR amplification wasperformed using the cDNA as templates and primers complementary to cDNA5′ and 3′ flanking sequence. The sequences of the primers used arepresented in Table 3. The amplified products were resolved on agarosegel electrophoresis.

TABLE 3 Primer sequences for RT-PCR Anealing Size of PCR Temp productGene Primer Sequence(5′→-3′) (° C.) (bp) Peroxisome forwardTTCGGAATCAGCTCTGTGGA 55 148 proliferator activated primer (SEQ ID NO: 1)receptor reverse CCATTGGGTCAGCTCTTGTG gamma(PPARγ2) primer(SEQ ID NO: 2) Fatty acid binding forward AGCATCATAACCCTAGATGG 55 128protein(aP2) primer (SEQ ID NO: 3) reverse GAAGTCACGCCTTTCATAAC primer(SEQ ID NO: 4) CCAAT/enhancer forward TCGGTGCGTCTAAGATGAGG 55 187binding protein primer (SEQ ID NO: 5) alpha(C/EBPα) reverseTCAAGGCACATTTTTGCTCC primer (SEQ ID NO: 6) TNFalpha(TNFα) forwardTGTCTCAGCCTCTTCTCATT 55 156 primer (SEQ ID NO: 7) reverseAGATGATCTGAGTGTGAGGG primer (SEQ ID NO: 8) Intereukin-6(IL-6) forwardATGAAGTTCCTCTCTGCAAG 55 638 primer AGACT (SEQ ID NO: 9) reverseCACTAGGTTTGCCGAGTAGA primer TCTC (SEQ ID NO: 10) Glyceraldehyde-3-forward AGAACATCATCCCTGCATCC 55 321 phosphate primer (SEQ ID NO: 11)dehydrogenase reverse TCCACCACCCTGTTGCTGTA (GAPDH) primer(SEQ ID NO: 12)

2) Results of RT-PCR Analysis of Visceral Fat Tissues

The mRNA expressions of visceral fat tissues were evaluated by RT-PCRanalysis. The HFD group showed significantly increased expressions ofC/EBPα, PPARγ2 and aP2 genes as compared to the Chow group. Nucleartranscription factors, C/EBPα and PPARγ2 play an important role inadipogenesis and aP2 is the target gene for these transcription factors.In contrast, adipic acid supplemented to the HFD significantly decreasedthe expression of C/EBPα, PPARγ2 and aP2 genes to levels similar for theChow group (FIG. 8). Accordingly, it could be understood that adipicacid prevented HFD-induced accumulation of fat in the visceral adiposetissue by decreasing the expression of nuclear transcription factors andtheir target gene.

It was well known to one of skill in the art that diet-induced obesityanimal models or obese humans exhibit Type 2 diabetes showingsimultaneously elevated blood insulin and glucose levels in fastedstate. A term “metaflammation” was recently coined to indicateinflammations induced by excess supply of nutrients or metabolites andobesity was indicated as chronic and low-level inflammation,highlighting the correlation between obesity and immune system. Forexample, the TLR4 (toll-like receptor 4) molecule responsible for innateimmune responses plays a pivotal role in inflammation and insulinresistance pathway in response to dietary fats (particularly, saturatedfatty acids) as ligands.

When obesity is induced by HFD, the free fatty acids (especiallysaturated fatty acids) in body fluids are increased. The free fattyacids as ligands bound to TLR4 activate IKK and then NF-κB, andstimulate the secretion of pro-inflammatory cytokines such as TNF-α andIL-6 to cause inflammatory response. In addition, TNF-α and IL-6activate both the cytokine signaling 3 (SOCS3) and JNK and inducephosphorylation of serine residues of insulin receptor substrates (IRS)to inhibit glucose transport, finally causing insulin resistance inperipheral tissues of liver or muscle.

The mRNA expression profiles of pro-inflammatory cytokines in visceralfat tissues were evaluated using RT-PCR. The HFD group exhibitedsignificantly higher levels of both TNF-α and IL-6 gene expressions ascompared to the Chow group. Meanwhile, these HFD-induced elevations inthe expression of TNF-α and IL-6 were significantly reversed by feedingadipic acid (FIG. 9). Accordingly, it would be concluded that adipicacid has the excellent effects of decreasing inflammatory responses inthe visceral fat tissues.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present disclosure. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the disclosure as set forthin the appended claims.

What is claimed is:
 1. A method for treating a metabolic disease, themethod comprising administering to a mammalian subject in need thereofan effective amount of a food composition comprising adipic acid,wherein the metabolic disease is obesity, diabetes, dyslipidemia orfatty liver.
 2. The method according to claim 1, wherein the foodcomposition decreases an amount of blood fat, liver fat or visceral fatin the mammalian subject.
 3. The method according to claim 2, whereinthe blood fat, liver fat or visceral fat comprises triglyceride,cholesterol and free fatty acid.
 4. The method according to claim 3,wherein the visceral fat is one or more selected from the groupconsisting of epididymal fat, perirenal fat, mesenteric fat andretroperitoneal fat.
 5. The method according to claim 1, wherein thefood composition decreases activity of ALT (alanine aminotransferase) orAST (aspartate aminotransferase) in the mammalian subject.
 6. The methodaccording to claim 1, wherein the food composition decreases expressionof PPARγ (Peroxisome proliferator activated receptor gamma), C/EBPs(CCAAT enhancer-binding proteins) or aP2 (fatty acid binding protein) inthe mammalian subject.
 7. The method according to claim 1, wherein thefood composition decreases expression of TNF-α (tumor necrosisfactor-alpha) and IL-6 (interleukin-6) in the mammalian subject.
 8. Themethod according to claim 1, wherein the food composition decreases aglucose level in blood of the mammalian subject.
 9. The method accordingto claim 1, wherein the adipic acid is derived from plants.